Substrate-Independent Layer-By-Layer Assembly Using Catechol-Functionalized Polymers

ABSTRACT

The present invention provides a simple, non-destructive and versatile method that enables layer-by-layer (LbL) assembly to be performed on virtually any substrate. A novel catechol-functionalized polymer which adsorbs to virtually all surfaces and can serve as a platform for LbL assembly in a surface-independent fashion is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/622,136filed Sep. 18, 2012, which is a continuation of U.S. application Ser.No. 12/267,822 filed Nov. 10, 2008 and issued as U.S. Pat. No. 8,293,867on Oct. 23, 2012, which claims the benefit of U.S. ProvisionalApplication No. 60/986,847 filed Nov. 9, 2007. Each of theseapplications is hereby incorporated by reference in its entirety for allpurposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. DE014193awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Layer-by-layer (LbL) assembly allows one to create multifunctional filmson surfaces while maintaining the bulk properties of the individualsurfaces [1]. The method relies on sequential adsorption of polymersonto bulk surfaces from solution, giving rise to complexmultifunctional, multilayered films. LbL assembly is simple to implementand offers extensive control over film properties and composition duringstepwise adsorption of components.

Although the vast majority of LbL films are built from polyelectrolytesvia electrostatic interaction between layers, more recently LbL filmshave been made with hydrogen bonding of polymers [2], and other buildingblocks such as inorganic nanoparticles, giving access to even greatercontrol of chemical and physical properties of LbL films.

In principle, LbL assembly can be performed on a wide variety ofsubstrates, including noble metals (Au, Pt, etc.), oxides (quartz, Si,TiO₂, mica etc.), and synthetic polymers (polyethylene terephthalate(PET), poly(methyl methacrylate) (PMMA), polyetherimide, etc.) [3,4]. Inpractice, however, forming well-ordered LbL layers on many polymericsurfaces has proven challenging [5-7], and LbL assembly on hydrophobicpolymers such as poly(tetrafluoroethylene) (PTFE), and polyethylene (PE)often requires aggressive ‘priming’ methods such as plasma treatments[5,7], oxidative chemical reactions (piranha/persulfonation) [8,9], orpolymeric adsorption [6,10,11].

Accordingly, a need exists for catechol-functionalized polymers for usein LbL assembly of multifunctional films wherein the polymers allowsubstrate-independent LbL assembly.

SUMMARY OF THE INVENTION

The present invention provides novel methods and catechol-functionalizedpolymers for use in substrate independent layer-by-layer assembly ofmultifunctional films.

In one embodiment, the invention provides a catechol-functionalizedpolymer comprising at least 1 to 70 percent catechol functional groups.In a preferred embodiment, the polymer comprises 50 to 65 percentcatechol functional groups, and may comprise any anionic or cationicpolymer.

In a second embodiment, the invention provides catechol-functionalizedpolymer comprising the structure:

wherein “n” has a value in the range of from 10 to 10,000 and “m” has avalue in the range of from 1 to 5,000. Preferably the polymer has amolecular weight of at least 25 kDa and comprises at least 20 to 60percent catechol functional groups.

In a third embodiment, the invention provides a catechol-functionalizedpolymer comprising the structure:

wherein “x” has a value in the range from 10 to 10,000 and “y” has avalue in the range from 1 to 5,000. In a preferred embodiment, “x” is221 and “y” is 122.

In a fourth embodiment, the invention provides a catechol-functionalizedpolymer comprising the structure:

wherein “n” has a value in the range from 10 to 10,000 and “x” has avalue in the range from 1 to 5,000.

In a fifth embodiment, the invention provides a substrate-independentmethod for the layer-by-layer assembly of a multifunctional film. Themethod comprises the steps of: (a) contacting a substrate with a firstcatechol-functionalized polymer to form a first layer; (b) rinsing thesubstrate with neat solvent; (c) contacting the substrate with a secondcatechol-functionalized polymer to form a second layer; (d) rinsing thesubstrate with neat solvent, wherein steps (a) through (d) are repeateduntil a multifunctional multilayer film is formed.

In a preferred embodiment, the first catechol-functionalized polymercomprises the structure:

wherein “n” has a value in the range from 10 to 10,000 and “m” has avalue in the range of from 1 to 5,000.

In a further preferred embodiment, the second catechol-functionalizedpolymer comprises the structure:

wherein “x” has a value in the range from 10 to 10,000 and “y” has avalue in the range from 1 to 5,000.

In a sixth embodiment, the present invention provides asubstrate-independent method for the layer-by-layer assembly of amultifunctional film comprising the steps of: (a) contacting a substratewith a first catechol-functionalized polymer to form a first layer; (b)rinsing the substrate with neat solvent; (c) contacting the substratewith a second substance having an affinity for the first layer to form asecond layer; (d) rinsing the substrate with neat solvent, wherein steps(a) through (d) are repeated until a multifunctional multilayer film isformed.

Preferably, the first catechol-functionalized polymer comprises thestructure:

wherein “n” has a value in the range from 10 to 10,000 and “m” has avalue in the range of from 1 to 5,000.

Advantageously, the present invention provides novelcatechol-functionalized polymers for use in novel methods ofsubstrate-independent LbL assembly of multifunctional films. The novelpolymers of the present invention enable a simple, non-destructive andversatile method that enables LbL assembly to be performed on virtuallyany substrate. The invention provides a novel approach tosubstrate-independent LbL assembly by exploiting the strong interfacialbinding property of novel catechol-functionalized polymers.

The polymers and methods of the present invention avoid the need foraggressive chemical or physical pre-treatment regimens of substratesnormally required for LbL on challenging substrates such as neutral andhydrophobic polymers. The inventors have found that augmenting thenumber of catechol groups in a polymer structure provides superioradhesive strength and water resistance while maintaining the uniqueproperties of the unmodified polymer.

The novel catechol-functionalized polymers of the present inventionadsorb to virtually all surfaces and can serve as a platform for LbLassembly in a surface-independent fashion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Synthesis of catechol-containing polymers. (A)Catechol-functionalized Polyethyleneimine (PEI-C). (B)Catechol-functionalized Hyaluronic Acid (HA-C).

FIG. 2. Synthesis of catechol-functionalized poly(allylamine)(PAA-DHPA).

FIG. 3. Layer-by-layer assembly on PTFE. (A) XPS spectra of bare PTFE(top), after the first cycle assembly of PEI-C/HA-C (middle), afterthree cycles (bottom). (B) Surface composition of fluorine (F1s) as afunction of the number of LbL deposition cycles of PEI-C/HA-C. (C-E)Wetting of water on bare PTFE (C, θ_(stat)=106°), PTFE after threecycles of LbL assembly using PEI-C and HA-C (D, θ_(stat)=19.7°), andPTFE after three cycles assembly using PEI and HA (E, θ_(stat)=55.4°).

FIG. 4. Substrate-independent LbL assembly using PEI-C and HA-C. (A) XPSsurface nitrogen composition on various organic polymer surfaces afteradsorption of PEI (black) or PEI-C (gray). (B) Ellipsometric polymerfilm thickness versus number of cycles of PEI-C/HA-C adsorption onSiO_(x) (circles), Au (triangles), and PMMA (squares).

FIG. 5. Layer-by-layer assembly of PAA and PLL on PEI-C primed SiO_(x).(A) XPS spectrum after single-step PEI-C adsorption on SiO_(x) (B) XPSspectrum of (PEI-C/PAA)₁-(PLL/PAA)₁₀ adsorption on SiO_(x). (C)Ellipsometry thickness of (PEI-catechol/PAA)₁-(PLL/PAA)_(n). AFM imageof a bare SiOx substrate (D), after (PEI-C/PAA)₁ deposition (E), andafter (PEI-C/PAA)₁-(PLL/PAA)₁₀ deposition (F). AFM images showedrelatively smooth topography of the polymeric deposition.

FIG. 6. Catechol-mediated silver nanoparticle formation in LbL films ofPEI-C/HA-C and antibacterial activity of the nanocomposite films. (A)Schematic illustration of Ag nanoparticle formation in LbL film viacatechol oxidation in the presence of Ag(III). (B-D) Topographic AFMimages of the LbL film after PEI-C/HA-C (n=20) deposition (B), and thesame film incubated in 1 mM AgNO₃ solution for 30 min (C) and 18 hrs(D). (E) XPS spectra of the silver incorporated LbL film shown in D (18hrs). Metallic silver photoelectron (3d_(5/2)) was detected at thebinding energy of 368.4 eV. (F). Live-dead assay of adhered E. coli onbare Si, LbL (n=20), and LbL+Ag (n=20, 18 hrs) surfaces.

FIG. 7. Transmission electron microscopy images of TiO₂ nanosheets.

FIG. 8. Bilayer thickness as measured by ellipsometry for ½ bilayer, 1bilayer, 2 bilayers and 4 bilayers on a Ti-coated substrate (triangles)and Si wafer (x's). In both cases, the trendline has a coefficient ofdetermination above 0.99.

FIG. 9. EDS Spectra of Si wafer after ½ bilayer of PAA-DHPA (top) andafter 4 bilayers of PAA-DHPA and TiO₂ nanosheets. At bilayer 4, note theC, O and Ti peaks in our sample.

FIG. 10. EDS spectra of Ti-coated Si wafer after ½ bilayer of PAA-DHPA(top) and after 1 bilayer of PAA-DHPA and TiO₂ nanosheets. Due to thepenetrative nature of EDS that the Si peak dominates thecharacterization and Ti is only detected at 1 bilayer.

DETAILED DESCRIPTION I. In General

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . . ” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, “characterized by” and “having” can beused interchangeably.

This invention is not limited to the particular methodology, protocols,and reagents described, as these may vary. The terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.

II. The Invention

The present invention provides novel catechol-functionalized polymersfor use in layer-by-layer assembly of multifunctional films and methodsof use thereof.

Catechol-Functionalized Polymers. The present invention provides novelcatechol-functionalized polymers for use in layer-by-layer assembly ofmultifunctional films. By “catechol-functionalized” we mean modifying apolymer to increase the number of catechol functional groups existing inthe polymer structure. By “catechol functional group” we mean theorganic compound with the formula:

where R1, R2, R3 and R4 may be hydrogen, an organic group including butnot limited to such groups as an alkyl or aryl group, or a carboxylateor sulfonate group. In an alternative embodiment, the polymer may alsobe functionalized with pyrogallol, where 3 OH groups are on an aromaticring in the 3,4,5 or 2,3,4 positions.

The resulting modified polymer structure includes an additional numberof covalently bonded catechol functional groups beyond those which mayalready exist in the unmodified polymer structure. In particular, theadditional catechol groups are introduced in terminal and/or pendantpositions in the polymer structure. As a result of their terminal and/orpendant position, the additional catechol groups are free for substrateadhesive bonding and crosslinking. These additional free functionalgroups typically are covalently bonded to the polymer via linking groupssuch as amides, ethers, urethanes or esters.

The catechol-functionalized polymers of the present invention preferablycontain at least 20 to 30 weight percent additional catechol functionalgroups. However, the polymer may be modified to incorporate anywherefrom about 0.1 weight percent to about 90 weight percent additionalcatechol groups, based on the total weight of the modified polymer. In apreferred version, the polymer may be modified to incorporate about 1 to70 weight percent additional catechol groups. In a further preferredversion, the polymer may be modified to incorporate about 2 to 50 weightpercent catechol groups.

The catechol-functionalized polymers of the present invention can be ofany size, although preferably, x+y=10 to 10000 and y=1-90% of total(x+y). The catechol-functionalized polymers of the present inventionalso may have a molecular weight in the range of 5 to 1,000,000 kDa,although in a preferred version the polymers have a molecular weight of20 to 500 kDa.

While the present application describes the synthesis of threecatechol-functionalized polymers, catechol-functionalizedpoly(ethylenimine) (PEI-C), catechol-functionalized hyaluronic acid(HA-C) and catechol-functionalized poly(allylamine) (PAA-DHPA), anypolymers useful in LbL assembly may be functionalized for use in thisinvention, including but not limited to any polycation or polyanion ofbiological, semi-synthetic or synthetic origin.

Methods of Use. The present invention also provides novel methods ofsubstrate-independent Layer-by-Layer (LbL) assembly of multifunctionalfilms using at least one of the catechol-functionalized polymers of thepresent invention.

In use, LbL assembly is carried out in a conventional manner accordingto the following steps. One, a substrate is dipped in a firstcatechol-functionalized polymer. Two, the substrate is rinsed in neatsolvent, such as deionized water, methanol or other suitablecompositions until substantially free of the catechol-functionalizedpolymer being applied. Three, the substrate is dipped in a secondsubstance, the second substance having an affinity for the firstsubstance. The second substance may be a second catechol-functionalizedpolymer of the present invention or another substance having an affinityfor the first catechol- functionalized polymer. Finally, the substrateis rinsed again in neat solvent. These steps are repeated in a cyclicfashion until the desired number of layers have been deposited on thesubstrate. The desired number of layers is achieved when the requiredthickness or the desired properties are achieved.

As used herein, one substance can be said to have an affinity foranother substance via either an electrostatic attraction or by virtue ofvan der Waals' forces, hydrogen bond forces, electron exchange or othertypes of chemical interactions.

For instance, in a preferred embodiment, the firstcatechol-functionalized polymer comprises a positively chargedpolyelectrolyte. The electrostatic attraction between thepolyelectrolyte and the substrate results in the adsorption of a layerof polyelectrolyte to the substrate. The second substance is preferablya negatively charged material such as, by way of example and notlimitation, polyelectrolyte, polymers, proteins, dyes, metal andsemiconductor nanoparticles, magnetic nanoparticles, vesicles, viruses,DNA, RNA and the like.

Substitutions of polymers/substances with a like charge or affinity maybe made for the first and second substances to achieve the sequentialadsorption of layers of a plurality of polymers/substances resulting indesired properties.

The sequence of the layers is determined by the order of dipping. Thepolymers/substances adsorbed at various layers may be easily varied suchthat layers of different materials can be combined depending on therequired functionality or combination of functions required. Thus,sequential adsorption of monolayers of polyelectrolytes, dyes,nanoparticles (metal, semiconducting, magnetic, etc.), polymers,proteins, vesicles, viruses, DNAs, RNAs, oligonucleotides, organic andinorganic colloids and other substances on layers of, for example, apolyelectrolyte having an affinity therefore, allows for theunprecedented control over film structure, production of multifunctionalmembranes, incorporation of biological compounds into the film whileretaining their biological activity, and improvement of the performanceof the film in most applications.

Substrate Independent. The catechol-functionalized polymers of thepresent invention facilitate LbL assembly on a wide variety ofsubstrates, a strategy referred to by the inventors assubstrate-independent layer-by-layer (siLbL) assembly. For instance, inaddition to the traditional substrates commonly used in LbL assembly,the catechol-functionalized polymers of the present invention allow LbLassembly on substrates including but not limited to metal, oxide andpolymer substrates.

For instance, a large number of medical devices are made out ofpoly(tetrafluoroethylene), and this material is traditionally difficultto surface modify. The polymers and methods of the present invention canbe used to provide surface modification of devices made withpoly(tetrafluoroethylene) for such thing as incorporating antibioticdrugs or silver nanoparticles for enhancing bacterial resistance ofmedical devices, or incorporating DNA onto device surfaces for purposesof gene therapy. In this case the DNA itself can be employed as apolyanion in the LbL strategy. The LbL films can have nonmedical usesalso, such as for example acting as adhesion promoters for applyingother coatings to inert or typically difficult surfaces likepolyethylene, etc.

Universal Primer. PEI-C also functions as a universal primer tofacilitate subsequent LbL with other polymers. The inventorsdemonstrated this concept on a silicon wafer with poly(acrylic acid)(PAA) and poly-L-lysine (PLL), two polymers that have a history of usein LbL assembly [19].

Enhanced Function. Certain functional properties of LbL films may beenhanced by incorporation of catechol residues into LbL films. Forexample, the strong interaction of catechols with surfaces [20,21]suggests that LbL films deposited onto a primer layer of PEI-C shouldenhance adhesion and help prevent delamination of LbL films fromsubstrate surfaces. Likewise, catechols could enhance mechanicalproperties within LbL composite films. Accordingly, thecatechol-functionalized polymers of the present invention demonstrate auseful functional property of LbL multilayer films constructed from saidcatechol-containing polymers.

Reducing Agent. The catechol groups in the LbL film are redox active andtherefore can function as a reducing agent to oxidize metal ions, aspreviously demonstrated for spontaneous electroless Ag and Cumetallization of catecholamine polymer coated surfaces from aqueousmetal salt solutions [14]. By utilizing the latent reactivity ofcatechol functional groups in the catechol-functionalized polymers ofLbL films for in-situ reduction of metal ions within the LbL multilayerfilm, the functionality of the LbL film can be designed to impart, forinstance, an antibacterial property to the multilayer film. In addition,oxidation of catechol-containing LbL films through treatment with achemical or enzymatic oxidizer, or by use of alkaline pH, can be used tocovalently cross-link catechols to each other or to other functionalgroups, which in turn can improve the rigidity and strength of the LbLfilm.

Kits. In an alternate embodiment of the invention, a kit for preparingthe polymers of the present invention is provided. In one embodiment,the kit comprises at least one of the catechol-functionalized polymersof the present invention and instructions for use.

By “instructions for use” we mean a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the invention for one of the purposes set forth herein.The instructional material of the kit can, for example, be affixed to acontainer which contains the present invention or be shipped togetherwith a container which contains the invention. Alternatively, theinstructional material can be shipped separately from the container orprovided on an electronically accessible form on a internet website withthe intention that the instructional material and the biocompatiblehydrogel be used cooperatively by the recipient.

The following examples are, of course, offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and the followingexamples and fall within the scope of the appended claims.

III. Examples Example 1 Synthesis of catechol-Functionalized Polymers

Catechol-functionalized Polyethylenimine (PEI-C). Poly(ethylenimine)(PEI), a cationic polymer with a history of use in LbL assembly [10,15],was conjugated with 3-(3,4-dihydroxyphenyl)propionic acid to makecatechol-functionalized PEI (PEI-C) (FIG. 1A). The degree of catecholmodification in PEI-C was 63% as determined by the ninhydrin test,thereby preserving the cationic character of the polymer for use in LbLwhile at the same time mimicking the high catechol content of musseladhesive proteins [12].

3 g of PEI (M_(w)=25 ikDa, Sigma-Aldrich) was dissolved in 300 ml of PBSsolution adjusted to pH 5.5 using 1 N HCl solution. 1.52 g (17.4 mmol)of 3-(3,4-dihydroxyphenyl) propionic acid and 2.71 g (34.9 mmol) of EDCwere added, and the pH of the reaction solution was maintained at 5.5for 2 hour with 1.0 N NaOH. Unreacted chemicals and urea byproducts wereremoved by extensive dialysis. Degree of substitution was determined byninhydrin test.

Catechol-functionalized Hyaluronic Acid (HA-C). Hyaluronic acid (HA) isa linear polysaccharide found in the extracellular matrix (ECM) ofconnective tissues and has been used in LbL assembly [16,17]. Acatechol-modified HA was synthesized by reacting dopamine with HA in thepresence of EDC, yielding HA-catechol (HA-C) with 35.6% of carboxylgroups modified by dopamine (FIG. 1B).

1 g of HA (M_(w)=130 kDa, Lifecore) was dissolved in 100 ml of PBSsolution adjusted to pH 5.5 using 1 N HCl solution. 388.1 mg (2.5 mmol)of EDC and 474.1 mg (2.5 mmol) of dopamine hydrochloride were added, andthe pH of the reaction solution was maintained at 5.5 for 2 hours with1.0 N NaOH. This reaction resulted in modification of 35.6% of primaryamine groups.

Catechol-functionalized Polyallylamine (PAA-DHPA). Polyallylamine (PAA)is a cationic polyelectrolyte that can be used in combination with ananionic polyelectrolyte like poly(sodium styrene sulfonate) to form alayer-by-layer adsorbed film of negatively and positively chargedpolymers.

5 g PAA (53 mmol monomer units, Mw=65 kDa, Sigma-Aldrich) was dissolvedin 100 mL anhydrous methanol. Upon addition of potassium hydroxide (2.9g, 53 mmol), a white precipitate appeared and was removed by filtrationand the filtrate was added with dry DMF, followed by reduction ofmethanol by rotary evaporation and addition of NHS ester of acetonideprotected 3-(3,4-dihydroxyphenyl)propanoic acid (DHPA-NHS) (1.7 g, 5.3mmol). The mixture was stirred under argon protection for one day. Afterthe volatile was reduced by vacuum, cold dry ether was added to producea white precipitate, which was collected by centrifuge. The obtainedsolid was subject to acetonide-deprotection in 100 mL ofDMSO/TFA/TIS/water (20:70:2.5:7.5) overnight to yield catechol graftedpolyallylamine The modification degree was estimated by ¹H NMR and couldbe controlled by the molar ratio of DHPA-NHS and poly(allylamine)monomer units.

Example 2 Layer-by-Layer Assembly

In this example, the inventors demonstrate the Layer-by-Layer (LbL)assembly of films on various substrates.

PTFE, PE, PC, PET, PMMA, Si, and Au surfaces were ultrasonically cleanedin deionized water for 5 min and transferred to the PEI-C and HA-Csolutions (5 mg/mL in water, pH 6.5) for LbL assembly. The followingcycle was generally used: (1) PEI-C for 3 min, (2) wash in water for 1min, (3) HA-C for 3 min, and (4) wash in water for 1 min. For PTFE, thefirst PEI-C/HA-C adsorption was carried out for 2 hrs, and subsequentsteps were same as described. A control experiment involving LbL on PTFEusing as-supplied PEI (no catechol) in each assembly step was performedwith overnight adsorptions (18-24 hrs). The same method was used forheterogeneous assembly of PEI-C/PAA (M_(w)=90 kDa, Polysciences)followed by alternating PLL/PAA adsorption. Concentrations of PAA andPLL (Ave M_(w)=28,000 Da, Sigma-Aldrich) were 3 mg/mL in 10 mM Tris, pH7.0.

Characterization. Spectroscopic ellipsometry (Woollam Co., Inc. Lincoln,Nebr.) was used to determine the film thickness. AFM surface topographywas measured in air using an MFP-3D atomic force microscopy (AsylumResearch, San Diego, Calif.) operated in AC and contact modes. X-rayphotoelectron spectroscopy (Omicron ESCALAB) (Omicron, Taunusstein,Germany) was performed to measure surface atomic composition. XPS isconfigured with a monochromated Al Ka (1486.8 eV) 300-W X-ray sourcewith an ultrahigh vacuum (<10⁸ Torr). The takeoff angle was fixed at45°, and all spectra were calibrated using the hydrocarbon C(1 s) peak(284.5 eV).

Bactericidal testing. E. coli (ATCC 35218) was grown in MHB(Mueller-Hinton Broth, cation adjusted) at 37° C. for 24 h frompreviously frozen inoculums. Substrates were sterilized by UV treatmentand incubated at 37° C. with 1 mL of phosphate buffered saline (PBS)containing ˜10⁵ CFU/mL E. coli for 4 hrs with mild agitation. Substrateswere rinsed with PBS and stained with Syto 9 and propidium iodide in PBS(2 uL/mL) for 10 min and then mountained on glass slides. Attachedbacteria were imaged using a Leica epifluorescence microscope (40× mag).

Example 3 LbL Assembly on PTFE

The inventors first demonstrated LbL assembly on PTFE, an example of aparticularly challenging substrate for LbL due to its anti-adhesiveproperty [18]. The progress of LbL assembly was monitored by X-rayphotoelectron spectroscopy (XPS) as shown in FIG. 3. The intensity offluorine 1s (F1s) (690 eV) and carbon 1s (C1s) (292 eV, C-F) peaks frombare PTFE (FIG. 3A, top) decreased after the first cycle of PEI-C/HA-Cassembly (FIG. 3A, middle) and completely disappeared after only threecycles (FIG. 3A, bottom). The fluorine composition at the PTFE surfacedecreased from 69 percent initially to only 1.6 percent after two-cyclesof PEI-C/HA-C assembly (FIG. 3B), demonstrating well-controlled LbLdeposition on untreated PTFE. Contact angle measurements clearly showedthe stark contrast in wetting characteristics of the PTFE surface beforeand after LbL assembly (FIG. 3C-D); the advancing contact angle(θ_(adv)) decreased from 115° for unmodified PTFE to 27.8° afterthree-cycle assembly (PEI-C/HA-C)₃. The importance of the catecholfunctionality in effective LbL on PTFE was illustrated by poor wetting(θ_(adv)=69.5°) when unmodified PEI and HA were used under the conditionof significantly extended adsorption times (18˜24 hrs per each assembly)(FIG. 3E).

Example 4 LbL Assembly on Noble Metals, Oxides and Polymer Substrates

In this example, the inventors demonstrate the substrate versatility ofthe novel catechol-functionalized polymers of the present invention.

LbL assembly on a variety of organic and inorganic surfaces wasfacilitated using alternating cycles of PEI-C/HA-C adsorption. Theinventors used Au, SiO_(x), and PMMA as representatives of noble metal,oxide, and polymer substrates, respectively. Ellipsometric measurementof film thickness resulting from PEI-C/HA-C adsorption revealed a filmdeposition rate of 2.1 nm/cycle (n) regardless of substrate (FIG. 4B).LbL assembly was also performed on several other polymeric surfaces (PE,PET, and polycarbonate (PC)) generally considered to be difficult tofunctionalize without prior surface modification. Comparative XPSstudies of PEI vs. PEI-C adsorption on these substrates confirmed theimportance of catechol residues in first layer adsorption. For example,the nitrogen signal (N1s), a useful indicator due to its presence inPEI-C chains but not in the substrate, showed that PE wasanti-adsorptive to PEI but was readily modified by PEI-C. On PET and PC,trace amounts of nitrogen were detected following adsorption of PEI,although the nitrogen amount was higher when PEI-C was used.Quantitative XPS analysis of surfaces modified by PEI-C all containedsimilar nitrogen levels (5-7 percent) regardless of substrate, whereasPEI modification of the same surfaces yielded uniformly low nitrogencontent (0-2 percent) (FIG. 4A).

Example 5 In-situ Reduction of Metal Ions Within the LbL Multilayer

In this example, the inventors demonstrate that thecatechol-functionalized polymers of the present invention can be usedfor the in situ reduction of metal ions.

Specifically, the latent reactivity of catechol functional groups inPEI-C/HA-C LbL films was used for in-situ reduction of Ag(III) to Ag(0)within the LbL multilayer (FIG. 6A). First, LbL films of PEI-C/HA-C(n=20) were assembled on SiO_(x). Subsequently, the LbL film andsubstrate were transferred to a silver nitrate solution (1 mM), uponwhich AFM imaging of the surface revealed topological changescorresponding to Ag nanoparticle formation. XPS analysis indicated astrong signal at 368.4 eV (FIG. 6E), corresponding to the reportedbinding energy of metallic silver (3d_(5/2)) [23]. Given theantimicrobial activity of metallic silver [24], the bactericidal effectof the incorporated silver particles in the LbL film was tested in anin-vitro adhesion experiment with Escherichia coli. Surfaces wereinoculated with 10⁵ CFU of E. coli for four hours and then the number ofdead bacteria attached to the surface counted. Ag nanoparticle-embeddedLbL films showed enhanced anti-bacterial effects compared to the LbLfilm without Ag and the bare SiOx surface (FIG. 6F).

Example 6 Catechol-Functionalized Polymers as a Universal Primer

In this example, the inventors show that catechol-functionalizedpolymers of the present invention may be utilized as universal primersto facilitate subsequent LbL assembly with other polymers.

First, PEI-C was adsorbed as a primer layer on SiO_(x), after which XPSanalysis revealed peaks representative of both substrate (99.5 eV forSi2p, and 143 eV for Si2s) and polymer (285 eV for C1s and 400 eV forN1s) (FIG. 5A). The strong oxygen is (O1 s) peak at 535 eV containscontributions from the silicon oxide and hydroxyl groups of thecatechol. Adsorption of PAA followed by ten subsequent cycles of PLL/PAAadsorption [(PEI-C/PAA)₁-(PLL/PAA)₁₀] and XPS analysis resulted incomplete suppression of substrate signals (Si2p,2s), leaving only C1s,N1s and O1s peaks corresponding to PAA and PLL (FIG. 5B). The thicknessof the multilayer film was monitored by spectroscopic ellipsometryduring LbL assembly, revealing a roughly linear increase in thicknesswith PLL/PAA deposition (FIG. 5C). Atomic force microscopy (AFM) imagingrevealed a morphological transition from rough at an early stage touniform film formation after many layers (FIG. 5D-F). The change ofsurface morphology could influence contact angle measurements.

Example 7 Synthesis of TiO₂ Nanosheets for LbL Assembly

In this example the inventors demonstrate the synthesis of TiO₂nanosheets for use in LbL assembly.

Synthesis of TiO₂ nanosheets. The TiO₂ nanosheets were synthesizedaccording to literature [25]. To a 50 mL crucible were added a wellmixed mixture of Cs₂CO₃(8.5 g, Sigma-Aldrich) and TiO₂ nanoparticles(10.8 g). The crucible was heated at 800° C. for 24 h and cooled down toroom temperature. After re-mixing, the crucible was heated at 800° C.for another 24 h. A portion of the obtained powder (5 g) was treatedwith 150 mL 1N HCl solution. The mixture was shaken for a week on aflask shaker (St. John Associates) with renewal of the acid solutionevery 24 h. After filtration and wash with water, a portion of theresulting protonic titanate powder (1 g) was treated with 0.017 mol/Ltetrabutylammonium hydroxide (Sigma-Aldrich). The mixture was shaken for2 weeks to completely delaminate the titanate structure. Thus obtainedopaque solution was set on the bench for one week. The upper suspensionwas decanted and used for the layer by layer coating. (The obtainedsuspension is quite stable at room temperature. There is only a littleamount of precipitate at the bottom over a period of one and a halfyears.) Transmission electron microscopy of TiO₂ nanosheets revealed thelamellar nature of the material (FIG. 7).

Example 8 LbL Assembly with TiO₂ Nanosheets

In this example the inventors provide a representative example of LbLassembly using PAA-DHPA and TiO₂ nanosheets to form a multilayerednanocomposite.

LbL Assembly. The inventors used Si wafers and Si wafers coated with 15nm thick layer of Ti by electron beam evaporation for the LbLsubstrates. The Si and Ti surfaces were ultrasonically cleaned inultrapure water (UP), 2-propanol, petroleum ether and then acetone.After drying under a stream of N₂, the wafers were further cleaned withoxygen plasma for 3 minutes at <150 Torr. The inventors created a 4mg/mL solution of PAA-DHPA in 50% dimethyl sulfoxide (DMSO) and 50% UPwater and used HCl to lower its pH from 7.5 to 2.9. The inventorsfurther created a 4 mg/mL solution of TiO₂ nanosheets in UP water. LBLwas performed by submerging the Si and Ti-coated wafers in alternatingsolutions of PAA-DHPA and TiO₂ nanosheets. Each 5-minute submersion wasfollowed by a 2-minute wash in 50% DMSO/50% UP water and 100% UP waterto remove excess polymer molecules and nanosheets before submersing inthe next solution. The inventors define a bilayer as one cycle ofpolymer and TiO₂ nanosheet adsorption so that ½ bilayer consists of onlypolymer.

Surface characterization. Modification of the Si wafers and Ti-coatedwafers with the LBL assembly of PAA-DHPA and TiO₂ nanosheets wasconfirmed by ellipsometry thickness measurements, contact anglemeasurements as well as elemental detection through Energy DispersiveX-ray Spectroscopy (EDS). Ellipsometer measurements were taken forsamples coated with ½ bilayer, 1 bilayer, 2 bilayers and 4 bilayers inour LBL technique. The mean thicknesses determined for each bilayer aredisplayed in FIG. 8. For LBL on the Si wafer, we found a mean bilayerthickness of 3.63 nm. For Ti-coated wafers, the inventors found a meanbilayer thickness of 5.41 nm. In both cases, the mean thickness of thefirst layer of PAA-DHPA was only 0.5 nm.

EDS spectra were captured for all LBL samples. In FIG. 9, the Sisubstrates with ½ bilayer is dominated by a strong peak for Si, asexpected. At 4 bilayers, the strong peak for Si is still present, butsignals for C and O were also detected due to the presence of PAA-DHPA.Signals for Ti indicate successful adsorption of Ti nanosheets on thesurfaces. In FIG. 10, a strong peak for Si dominates the Ti-coatedsubstrate with ½ bilayer. At 1 bilayer, Ti is detected presumably due tothe Ti coating and Ti nanosheets.

Advancing (θ_(adv)) and static contact angles were measured forultrapure water on the surfaces using an auto pipetting system(Rame´-Hart). The bare Si wafer had a static contact angle of67.0°+/−5.9° and an advancing contact angle of 65.1°+/−2.3°. With the4-cycle adsorption of titanium nanosheets, the static contact angleshifted to 37.6°+/−1.9° and the advancing contact angle shifted to45.1°+/−1.8°. This shift in the direction of lower contact angles isconsistent with the addition of hydrophilic TiO₂ nanosheets.Collectively, these results confirm the sequential adsorption of the LbLcomponents on Si and Ti-coated substrata.

It should be noted that the above description, attached figures andtheir descriptions are intended to be illustrative and not limiting ofthis invention. Many themes and variations of this invention will besuggested to one skilled in this and, in light of the disclosure. Allsuch themes and variations are within the contemplation hereof. Forinstance, while this invention has been described in conjunction withthe various exemplary embodiments outlined above, various alternatives,modifications, variations, improvements, and/or substantial equivalents,whether known or that rare or may be presently unforeseen, may becomeapparent to those having at least ordinary skill in the art. Variouschanges may be made without departing from the spirit and scope of theinvention. Therefore, the invention is intended to embrace all known orlater-developed alternatives, modifications, variations, improvements,and/or substantial equivalents of these exemplary embodiments.

REFERENCES

-   [1] G. Decher, J.-D. Hong, Makromol. Chem. Macromol. Symp. 1991, 46,    321.-   [2] J. L. Lutkenhaus et al., J. Am. Chem. Soc. 2005, 127, 17228.-   [3] P. T. Hammond, Adv. Mater. 2004, 16, 1271.-   [4] Z. Tang, Y. Wang, P. Podsiadlo, N. A. Kotov, Adv. Mater. 2006,    18, 3203.-   [5] D. E. Bergbreiter, Prog. Polym. Sci. 1994, 19, 529.-   [6] M. Raposo et al., Macromolecules 1997, 30, 6095.-   [7] M. C. Hsieh, R. J. Farris, T. J. McCarthy, Macromolecules 1997,    30, 8453.-   [8] G. Price, F. Keen, A. A. Clifton, Macromolecules 1996, 29, 5664.-   [9] H. Zhao et al., Langmuir 2007, 23, 1810.-   [10] A. Delcorte, P. Bertrand, E. Wischerhoff, A. Laschewsky,    Langmuir 1997, 13, 5125.-   [11] A. Khademhosseini et al., Adv. Mater. 2003, 15, 1995.-   [12] J. H. Waite, N. H. Andersen, S. Jewhurst, C. Sun., J. Adhesion    2005, 81, 1.-   [13] D. J. Crisp, G. Walker, G. A. Young, A. B. Yule, J. Coll.    Inter. Sci. 1985, 104, 40.-   [14] H. Lee, S. M. Dellatore, W. M. Miller, P. B. Messersmith.,    Science, 2007, 318, 426-   [15] C. Boura et al., Biomaterials 2003, 24, 3521.-   [16] C. Picart et al., Langmuir 2001, 17, 7414.-   [17] B. Thierry, F. M. Winnik, Y. Merhi, M. Tabrizian, J. Am. Chem.    Soc. 2003, 125, 7494.-   [18] J. Vicente et al., Acta Oto-Laryngol. 2006, 126, 144.-   [19] V. Pardo-Yissar, E. Katz, O. Lioubashevski, I. Willner.,    Langmuir 2001, 17, 1110.-   [20] H. Lee, N. F. Scherer, P. B. Messersmith, Proc. Nat. Acad. Sci.    USA 2006, 103, 12999.-   [21] H. Lee, B. P. Lee, P. B. Messersmith, Nature 2007, 448, 338.-   [22] P. Podsiadlo et al., Adv. Mater. 2007, 19, 949.-   [23] B. J. Murray et al., Nano Lett 2005, 5, 2319.-   [24] C. H. Ho, J. Tobis, C. Sprich, R. Thomann, J. C. Tiller, Adv.    Mater. 2004, 16, 957.-   [25] T. Sasaki et al., Chem. Mater. 2002, 14, 3524-3530.

We claim:
 1. A catechol-functionalized polymer comprising the structure:

wherein “x” has a value in the range from 10 to 10,000; and wherein “y”has a value in the range from 1 to 5,000.
 2. The catechol-functionalizedpolymer of claim 1 wherein “x” is 221 and “y” is
 122. 3. Acatechol-functionalized polymer comprising the structure:

wherein “n” has a value in the range from 10 to 10,000; and wherein “x”has a value in the range from 1 to 5,000.